PerOxynitrite, a biological oxidant formed from the reaction of Nitric Oxide (NO) with the superoxide radical, is associated with many Pathologies, including NeuroDegenerative Diseases, such as Multiple Sclerosis (MS).

Gout (HyperUricemic) and MS are almost mutually exclusive, and Uric Acid has therapeutic effects in mice with Experimental Allergic EncephaloMyelitis, an animal disease that models MS.

This evidence suggests that Uric Acid may scavenge PerOxynitrite and/or PerOxynitrite-derived reactive species. Therefore, we studied the kinetics of the reactions of PerOxynitrite with Uric Acid from pH 6.9 to 8.0.

Among the biological molecules in human Plasma whose rates of reaction with PerOxynitrite have been reported, CO2 is one of the fastest with a pseudo-first-order rate constant k(CO2)(/Plasma) = 46 s-1 (T = 37 degrees C, pH 7.4; assuming [CO2](Plasma) = 1 mM).

Thus PerOxynitrite reacts with CO2 in human blood Plasma nearly 920 times faster than with Uric Acid. Therefore, Uric Acid does not directly scavenge PerOxynitrite because Uric Acid can not compete for PerOxynitrite with CO2.

The therapeutic effects of Uric Acid may be related to the scavenging of the radicals CO*-3 and NO*2 that are formed from the reaction of PerOxynitrite with CO2.

We suggest that trapping secondary radicals that result from the fast reaction of PerOxynitrite with CO2 may represent a new and viable approach for ameliorating the adverse effects associated with PerOxynitrite in many diseases.

PerOxynitrite (ONOO-), a toxic product of the free radicals Nitric Oxide and SuperOxide, has been implicated in the PathoGenesis of CNS inflammatory diseases, including Multiple Sclerosis and its animal correlate Experimental AutoImmune EncephaloMyelitis (EAE).

In this study we have assessed the mode of action of Uric Acid (UA), a purine metabolite and ONOO- scavenger, in the treatment of EAE.

We show that if administered to mice before the onset of clinical EAE, UA interferes with the invasion of inflammatory cells into the CNS and prevents development of the disease.

In mice with active EAE, exogenously administered UA penetrates the already compromized Blood-CNS Barrier, blocks ONOO--mediated Tyrosine nitration and Apoptotic cell death in areas of inflammation in Spinal Cord tissues and promotes recovery of the animals.

Uric Acid, the naturally occurring product of Purine metabolism, is a strong Peroxynitrite scavenger, as demonstrated by the capacity to bind PerOxynitrite but not Nitric Oxide (NO) produced by LipoPolySaccharide-stimulated cells of a mouse Monocyte line.

In this study, we used Uric Acid to treat Experimental Allergic EncephaloMyelitis (EAE) in the PLSJL strain of mice, which develop a chronic form of the disease with remissions and exacerbations.

Uric Acid administration was found to have strong therapeutic effects in a dose-dependent fashion.

A regimen of four daily doses of 500 mg/kg Uric Acid was required to promote long-term survival regardless of whether treatment was initiated before or after the clinical symptoms of EAE had appeared.

The requirement for multiple doses is likely to be caused by the rapid clearance of Uric Acid in mice which, unlike humans, metabolize Uric Acid a step further to Allantoin.

Uric Acid treatment also was found to diminish clinical signs of a disease resembling EAE in Interferon-gamma receptor knockout mice.

A possible association between Multiple Sclerosis (MS), the disease on which EAE is modeled, and Uric Acid is supported by the finding that patients with MS have significantly lower levels of Serum Uric Acid than controls.

In addition, statistical evaluation of more than 20 million patient records for the incidence of MS and Gout (HyperUricemic) revealed that the two diseases are almost mutually exclusive, raising the possibility that HyperUricemia may protect against MS.

#5

Inosine Inhibits Inflammatory Cytokine Production By A PostTranscriptional Mechanism And Protects Against Endotoxin-Induced Shock

ExtraCellular Purines, including Adenosine and ATP, are potent endogenous ImmunoModulatory Molecules. Inosine, a degradation product of these Purines, can reach high concentrations in the ExtraCellular space under conditions associated with cellular metabolic stress such as Inflammation or Ischemia.

In the present study, we investigated whether ExtraCellular Inosine can affect Inflammatory/Immune processes.

In ImmunoStimulated Macrophages and Spleen Cells, Inosine potently inhibited the production of the ProInflammatory Cytokines TNF-, IL-1, IL-12, Macrophage-Inflammatory Protein-1, and IFN-γ, but failed to alter the production of the AntiInflammatory Cytokine IL-10.

The effect of Inosine did not require cellular uptake by Nucleoside Transporters and was partially reversed by blockade of Adenosine A1 and A2 receptors. Inosine inhibited Cytokine production by a PostTranscriptional mechanism.

The activity of Inosine was independent of activation of the p38 and p42/p44 Mitogen-activated protein kinases, the Phosphorylation of the c-Jun terminal kinase, the degradation of inhibitory factor kappaB, and elevation of IntraCellular cAMP.

Inosine suppressed ProInflammatory Cytokine production and mortality in a mouse EndoToxemic model. Taken together, Inosine has multiple AntiInflammatory Effects.

These findings, coupled with the fact that Inosine has very low toxicity, suggest that this agent may be useful in the treatment of Inflammatory/Ischemic Diseases.

#6

Therapeutic Intervention In EAE By Administration Of Uric Acid Precursors

Uric Acid (UA) is a purine metabolite that selectively inhibits PerOxynitrite-mediated reactions implicated in the pathogenesis of Multiple Sclerosis (MS) and other NeuroDegenerative Diseases.

Serum UA levels are inversely associated with the incidence of MS in humans because MS patients have low Serum UA levels and individuals with HyperUricemia (Gout) rarely develop the disease.

Moreover, the administration of UA is therapeutic in Experimental Allergic Encephalomyelitis (EAE), an animal model of MS. Thus, raising Serum UA levels in MS patients, by oral administration of a UA precursor such as Inosine, may have therapeutic value.

We have assessed the effects of Inosine, as well as Inosinic Acid, on parameters relevant to the chemical reactivity of PerOxynitrite and the pathogenesis of EAE.

Both had no effect on chemical reactions associated with PerOxynitrite, such as Tyrosine nitration, or on the activation of inflammatory cells in vitro.

Moreover, when mice treated with the Urate Oxidase Inhibitor Potassium Oxonate were fed Inosine or Inosinic Acid, Serum UA levels were elevated markedly for a period of hours, whereas only a minor, transient increase in Serum Inosine was detected.

Administration of Inosinic Acid suppressed the appearance of clinical signs of EAE and promoted recovery from ongoing disease.

The therapeutic effect on animals with active EAE was associated with increased UA, but not Inosine, levels in CNS tissue. We, therefore, conclude that the mode of action of Inosine and Inosinic Acid in EAE is via their metabolism to UA.

Several studies indicate that patients with Multiple Sclerosis (MS) have low Serum levels of the endogenous AntiOxidant Uric Acid (UA), although it has not been established whether UA is primarily deficient or secondarily reduced due to its Peroxynitrite scavenging activity.

We measured Serum Urate levels in 124 MS patients and 124 age- and sex-matched controls with Other Neurological Diseases.

In addition, we compared UA levels when MS patients were stratified according to disease activity (by means of clinical examination and MRI), duration, disability and course.

Our study favors the view that reduced UA in MS is a primary, constitutive loss of protection against Oxidative agents, which deserves further pathogenetic elucidation aimed at future therapeutic strategies.

The effect of ingesting some Purine-rich foods (Beef Liver, Haddock Fillets and Soybeans) on Uric Acid metabolism was investigated in 18 male subjects with no history of Gout or Kidney Disorder.

In a crossover design, three IsoEnergetic and IsoNitrogenous meals were fed to volunteers during a 3-week period. Only the content of Uricogenic bases (Adenine and HypoXanthine) varied among the test meals.

Ingestion of all experimental meals caused an increase in Serum Uric Acid levels at 120 minutes and this increase was more marked (about twofold) with Haddock and Soybean ingestion.

In all groups, the postprandial Serum Uric Acid levels at 240 minutes were lower than those obtained at 120 minutes, but still remained elevated in comparison to the fasting level.

The test foods had little or no effect on Serum and Urinary Creatinine values. As expected, 24-hour Urinary Uric Acid excretion was similar for the three test meals due to the IsoNitrogenous load of Proteins and Purines.

Assessment of each Purine base content rather than the total Purine content of foods should be considered in future recommendations for HyperUricemic individuals.

Uric Acid, an AntiOxidant, is reduced in Multiple Sclerosis (MS). Patients with Gout have a reduced incidence of MS. Optic Neuritis (ON), often the first manifestation of MS, is not known to be associated with reduced Uric Acid.

Patients with recent onset of ON were investigated to determine whether Uric Acid levels were reduced at presentation. Twenty-one patients with ON were included, 17 females and 4 males.

The mean (SD) Serum Uric Acid in the ON female group was 184.4 ( +/-55.1) micromol/L (range, 116-309 micromol/L), whilst in the control group it was 235.2 (+/- 50.2) micromol/L (range, 172-381 micromol/L).

The difference was statistically significant (chi2 = 8.93, P = 0.003). In the small male cohort, mean (SD) Serum Uric Acid was 305 (+/- 52.1) micromol/L, whilst in the control group it was 328 (+/- 80.4) micromol/L.

These differences were not statistically significant. Reduced AntiOxidant reserve is possibly an early pathogenic mechanism in Inflammatory DeMyelination, and raises the possibility that low Uric Acid levels could be an indicator of disease activity.

Since Optic Neuropathies of other causes were not investigated, future research needs to determine whether low Uric Acid represents a unique feature of Optic Neuritis or is seen in other Optic Neuropathies.

Background
A number of studies found that patients with Multiple Sclerosis (MS) have low Serum levels of Uric Acid. It is unclear whether this represents a primary deficit or secondary effect.

Uric Acid is a scavenger of Peroxynitrite, which is the product of Nitric Oxide (NO) and superoxide.

Because peripheral blood Leukocyte NO production and NO metabolites in serum are raised in MS patients, associations might be expected between Serum Uric Acid levels and peripheral NO production.

Methods
Serum levels of Uric Acid and NO production by peripheral blood Leukocytes were measured in 60 patients with MS without a relapse in the past 3 months, and 30 age- and sex-matched healthy controls.

Uric Acid was determined with the uricase PAP method, and NO production was assayed by measuring Nitrite concentration in supernatants of lysed Leukocytes.

There was no correlation between Leukocyte Nitrite concentration and Serum Uric Acid levels.

Conclusions
Our findings suggest that in MS patients there is no primary deficit in Serum Uric Acid. NO production by peripheral blood Leukocytes is increased, but there is no association with Serum Uric Acid levels.

Uric Acid (UA), a product of Purine metabolism, is a known scavenger of Peroxynitrite (ONOO(-)), which has been implicated in the pathogenesis of Multiple Sclerosis and Experimental Allergic Encephalomyelitis (EAE).

To determine whether the known therapeutic action of UA in EAE is mediated through its capacity to inactivate ONOO(-) or some other Immunoregulatory phenomenon, the effects of UA on Ag presentation, T-Cell reactivity, Ab production, and evidence of CNS inflammation were assessed.

The inclusion of physiological levels of UA in culture effectively inhibited ONOO(-)-mediated oxidation as well as Tyrosine nitration, which has been associated with damage in EAE and Multiple Sclerosis, but had no inhibitory effect on the T-Cell-proliferative response to Myelin Basic Protein (MBP) or on APC function.

In addition, UA treatment was found to have no notable effect on the development of the Immune response to MBP in vivo, as measured by the production of MBP-specific Ab and the induction of MBP-specific T-Cells

These findings are consistent with the notion that UA is therapeutic in EAE by inactivating ONOO(-), or a related molecule, which is produced by activated Monocytes and contributes to both enhanced Blood-CNS Barrier permeability as well as CNS tissue pathology.